Determination of thiamine and thiamine phosphates in excitable tissues as thiochrome derivatives by reversed-phase high-performance liquid chromatography on octadecyl silica

Bontemps, José; Philippe, Paul; Bettendorff, Lucien; Lombet, Jacques; Dandrifosse, Guy; Schoffeniels, E.; Crommen, Jacques

doi:10.1016/s0378-4347(00)84100-6

Cited by 42 publications

(14 citation statements)

References 23 publications

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“…1). Final concentrations were 2 ϫ 10 Ϫ4 mol/L for potassium ferricyanide and 5% for sodium hydroxide (13 ). After mixing, the plate was covered with a sealing tape and placed on the autosampler.…”

Section: Sample Preparationmentioning

confidence: 99%

Rapid HPLC Measurement of Thiamine and Its Phosphate Esters in Whole Blood

2008

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Section: Sample Preparationmentioning

confidence: 99%

Rapid HPLC Measurement of Thiamine and Its Phosphate Esters in Whole Blood

2008

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“…The quantification of thiamine, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) was based on the formation of thiochrome derivatives by alkaline oxidation (adapted from [34,35]). Briefly, perchloric cell or tissue extracts (100 μl; 0.4 M HClO 4 final) were derivatized with K 3 [Fe(CN) 6 ] (20 μl; 30.4 mM in 15% NaOH) after adding 10 μl methanol.…”

Section: Determination Of Thiamine and Its Phosphate Estersmentioning

confidence: 99%

Thiamine pyrophosphate: An essential cofactor for the α-oxidation in mammals – implications for thiamine deficiencies?

Sniekers

Foulon

Mannaerts

et al. 2006

Cell. Mol. Life Sci.

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The identification of 2-hydroxyphytanoyl-CoA lyase (2-HPCL), a thiamine pyrophosphate (TPP)-dependent peroxisomal enzyme involved in the alpha-oxidation of phytanic acid and of 2-hydroxy straight chain fatty acids, pointed towards a role of TPP in these processes. Until then, TPP had not been implicated in mammalian peroxisomal metabolism. The effect of thiamine deficiency on 2-HPCL and alpha-oxidation has not been studied, nor have possible adverse effects of deficient alpha-oxidation been considered in the pathogenesis of diseases associated with thiamine shortage, such as thiamine-responsive megaloblastic anemia (TRMA). Experiments with cultured cells and animal models showed that alpha-oxidation is controlled by the thiamine status of the cell/tissue/organism, and suggested that some pathological consequences of thiamine starvation could be related to impaired alpha-oxidation. Whereas accumulation of phytanic acid and/or 2-hydroxyfatty acids or their alpha-oxidation intermediates in TRMA patients given a normal supply of thiamine is unlikely, this may not be true when malnourished.

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“…Maximum reduction of FAD was achieved photochemically with the oxalate/K ϩ -5-deaza-10-methyl-3-sulfopropyl-isoalloxazine system (39); however, irradiation intervals of up to 30 min were required. Reoxidation of photochemically reduced CDH with aliquots of K 3 [Fe(CN) 6 ] proceeded via the formation of the blue neutral flavin radical with its typical absorbance at 575 nm to 625 nm (Fig. 2).…”

Section: Variation Of the Concentrations Of Cdo And Nadmentioning

confidence: 99%

“…The concentration of the semiquinone amounted to 6.2 M (ϳ20% of total FAD) based on the extinction coefficient ε 580-620 (ϳ4.00 mM Ϫ1 ⅐ cm Ϫ1 ) (37). The starting spectrum of the enzyme as isolated could not be fully restored (ϳ85%), even after the addition of an excess of K 3 [Fe(CN) 6 ] (4 to 10 equivalents per CDH monomer), most likely because of radiation damage to the FAD chromophore.…”

Section: Variation Of the Concentrations Of Cdo And Nadmentioning

confidence: 99%

“…Determination of thiamine. Samples were oxidized with 0.6 mM K 3 [Fe(CN) 6 ] in 14.7% NaOH (ratio, 2.6:1) to the corresponding thiochromes, which have a characteristic fluoresence ( ex ϭ 370 nm; em ϭ 444 nm [6]). They were analyzed on an HPLC system equipped with a diode array detector 400 and an SFM 25 fluorescence detector (Kontron Instruments), with a Gromsil C 18 reversed phase column that had been equilibrated with 25 mM KP i buffer, pH 8.4.…”

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Cyclohexane-1,2-Dione Hydrolase from Denitrifying Azoarcus sp. Strain 22Lin, a Novel Member of the Thiamine Diphosphate Enzyme Family

et al. 2011

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Alicyclic compounds with hydroxyl groups represent common structures in numerous natural compounds, such as terpenes and steroids. Their degradation by microorganisms in the absence of dioxygen may involve a COC bond ring cleavage to form an aliphatic intermediate that can be further oxidized. The cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) from denitrifying Azoarcus sp. strain 22Lin, grown on cyclohexane-1,2-diol as a sole electron donor and carbon source, is the first thiamine diphosphate (ThDP)-dependent enzyme characterized to date that cleaves a cyclic aliphatic compound. The degradation of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate comprises the cleavage of a COC bond adjacent to a carbonyl group, a typical feature of reactions catalyzed by ThDP-dependent enzymes. In the subsequent NAD ؉ -dependent reaction, 6-oxohexanoate is oxidized to adipate. CDH has been purified to homogeneity by the criteria of gel electrophoresis (a single band at ϳ59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (ϳ105 kDa; gel filtration). As isolated, CDH contains 0.8 ؎ 0.05 ThDP, 1.0 ؎ 0.02 Mg 2؉ , and 1.0 ؎ 0.015 flavin adenine dinucleotide (FAD) per monomer as a second organic cofactor, the role of which remains unclear. Strong reductants, Ti(III)-citrate, Na ؉ -dithionite, and the photochemical 5-deazaflavin/oxalate system, led to a partial reduction of the FAD chromophore. The cleavage product of CDO, 6-oxohexanoate, was also a substrate; the corresponding cyclic 1,3-and 1,4-diones did not react with CDH, nor did the cis-and trans-cyclohexane diols. The enzymes acetohydroxyacid synthase (AHAS) from Saccharomyces cerevisiae, pyruvate oxidase (POX) from Lactobacillus plantarum, benzoylformate decarboxylase from Pseudomonas putida, and pyruvate decarboxylase from Zymomonas mobilis were identified as the closest relatives of CDH by comparative amino acid sequence analysis, and a ThDP binding motif and a 2-fold Rossmann fold for FAD binding could be localized at the C-terminal end and central region of CDH, respectively. A first mechanism for the ring cleavage of CDO is presented, and it is suggested that the FAD cofactor in CDH is an evolutionary relict.Alicyclic compounds, such as steroids and terpenes, are widespread in nature. They are produced by plant cells as secondary metabolites and occur in fossil fuels. Microorganisms can convert these compounds to cellular metabolites under oxic and anoxic conditions. Their biodegradation proceeds via COC bond ring cleavage to form an aliphatic intermediate, which can be further degraded by ␤-oxidation. In aerobic bacteria, the cleavage of the cyclic compound is catalyzed by a NADPH-dependent, flavin-containing monooxygenase. For example, cyclohexanone is converted to ε-caprolactone in a Bayer-Villiger-type reaction (14). Subsequently, the lactone is hydrolyzed to 6-hydroxyhexanoate (63), followed by two NAD ϩ /NADP ϩ -dependent oxidation steps with adipate as the final product. In anaerobes, such as Pseudomonas sp. strain K601, cyclohexa...

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Determination of thiamine and thiamine phosphates in excitable tissues as thiochrome derivatives by reversed-phase high-performance liquid chromatography on octadecyl silica

Cited by 42 publications

References 23 publications

Rapid HPLC Measurement of Thiamine and Its Phosphate Esters in Whole Blood

Rapid HPLC Measurement of Thiamine and Its Phosphate Esters in Whole Blood

Thiamine pyrophosphate: An essential cofactor for the α-oxidation in mammals – implications for thiamine deficiencies?

Cyclohexane-1,2-Dione Hydrolase from Denitrifying Azoarcus sp. Strain 22Lin, a Novel Member of the Thiamine Diphosphate Enzyme Family

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